Tuberculosis is the number one killer disease. Every year it kills largest number of people due to a single infectious disease. According to a World Health Organization Report (WHO) over 8 million cases of tuberculosis are reported every year with over 2.9 million deaths
Tuberculosis deaths were gradually declining till early 90's by the virtue of availability of potent anti-tubercular drugs. The tuberculosis cases and deaths are again on rise mostly because of synergistic effect of co-infection with human immuno-deficiency virus (HIV)(Hopewell, P. C et al., 1992) and emergence of multiple drug resistant (MDR) strains of M. tuberculosis (Bloom, B. R, and C. L. Murray, 1992) and involvement of so called non tuberculous mycobacteria.
Over 70 species of mycobacteria are known, most are non-pathogenic for humans. Tuberculosis is caused by infection due to M. tuberculosis, with a few cases being caused by M. bovis. These organisms are genetically very close and called as Mycobacterium tuberculosis complex (MTC) organism. There is over a dozen other pathogenic mycobacteria, which causes tuberculosis like infection of lungs or other parts of the body. These organisms are called as mycobacteria other than tuberculosis (MOTT) or non-tuberculous mycobacteria (NTM). In the wake of AIDS epidemic these so called non tuberculous mycobacteria have become significant and are being isolated from large number of tuberculosis patients co-infected with HIV.
Early tuberculosis often goes unrecognized in an otherwise healthy individual. The lack of simple, rapid and reliable tests that can specifically detect M. tuberculosis and other causative agents in a clinical specimen poses enormous problems for both individual patient management and implementation of appropriate infection control and public health measures.
Classical methods of diagnosis include examination of a sputum smear under a microscope for acid-fast mycobactena and a X-ray of the lungs. However, in a vast majority of cases the sputum smear examination is negative for mycobacteria in the early stages of the disease, and lung changes may not be obvious on a X-ray until several months following infection. Staining of smear for acid-fast bacilli (AFB) takes less than two hours but lacks sensitivity and may be non-specific in some case. Moreover a positive result by AFB staining does not discriminate between the mycobacterium species.
Currently the only absolutely reliable method of diagnosis is based on culturing M. tuberculosis from the clinical specimen and identifying it morphologically and biochemically. Culturing of M. tuberculosis and other related organisms is sensitive and specific but is cumbersome and may take 6-12 weeks while culturing on solid media and three to six weeks on liquid media, during which time a patient may become seriously ill and infect other individuals. Therefore, a rapid test capable of reliably detecting the presence of M. tuberculosis is vital for the early detection, treatment and management of the patient.
Several molecular tests have been developed recently for the rapid detection and identification of M. tuberculosis. A commercial test, the Gen-Probe “Amplified Mycobacterium Tuberculosis Direct Test” has been evaluated by Abe et al and Miller et al. This test amplifies M. tuberculosis 16S ribosomal RNA from respiratory specimens and uses a chemiluminescent probe to detect the amplified product with a reported sensitivity of about 91%. Other commercial tests based on ligase chain reaction (LCR) (Abbott Laboratories), polymerase chain reaction (PCR) (Roche Diagnostics Systems, Eastman Kodak Co., Johnson & Johnson), Q-beta replicase (Gene Trak), and strand displacement amplification (Becton Dickinson).
Other methods based on immunological detection of infection with M. tuberculosis by non-culture methods are latex agglutination, radioimmunoassay and enzyme linked immunosorbent assays etc. Main drawback of these methods is their lack of sensitivity and or specificity (Kadival, et al., 1986; Yenez, et al., 1986). Serological techniques may be useful in some clinical settings but this approach is limited in general due to poor sensitivity and or specificity.
The development of polymerase chain reaction (PCR) (Saiki et al. 1988). that allows DNA to be amplified and detected from small amounts of nucleic acid samples has made it possible to detect M. tuberculosis specific nucleic acids in clinical specimens. Some of the earlier reports were based on the detection of the 16S ribosomal RNA or its gene. Detection of M. tuberculosis and related organisms by first amplifying a portion of DNA using a primer conserved for all bacteria then using species specific probes to detect different species of mycobacteria. Major drawback of this method is that this is cumbersome, and takes over 24 hours to complete. Species specific probes that are used to detect different species sequence in the amplified product vary only in few bases and subsequent analysis of this amplified DNA by assays based on hybridization, if carried out under less that ideal conditions can lead to a false positive test.
The discovery of the IS6110 insertion element (Thierry et. al. 1990) and the belief that this element may only be present in M. tuberculosis complex (M. tuberculosis, M. bovis, M. africanum and M. microti) spawned a whole series of rapid diagnostic strategies (Brisson-Noel et al., Clarridge et al., al., Forbes et al., Hermans et al., Kolk et al., Kox et al., Zambardi et al.). These tests employ various techniques to extract DNA from the sputum. PCR is used to amplify IS6110 DNA sequences from the extracted DNA. The successful amplification of this DNA is considered to be an indicator of the presence of M. tuberculosis infection. U.S. Pat. Nos. 5,168,039 and 5,370,998 has been issued to Crawford et al. for the IS6110 based detection of tuberculosis. Another U.S. Pat. No. 5,731,150 have been granted to CIBA CORNING DIAGNOSTICS CORP (Gurpreet. et.al). European patent EP 0,461,045 has been issued to Guesdon, J. L for the IS6110 based detection of tuberculosis. The IS6110 element was reported to be present at ten, two, one, five and five copies in M. tuberculosis, M bovis, M. bovis BCG, M. africanum and M. microti respectively. Most reports using IS6110 and other PCR based detection of tuberculosis claim sensitivities of over 75% and specificities approaching 100%.
A careful study on use of this sequence as a target for PCR based diagnosis of M. tuberculosis has reveled several drawbacks. A blind comparison study among 7 major laboratories authored by Noordhoek et al. raised a major concern when it reported false positive rates of 3 to 77% and sensitivities ranging from 2 to 90%. This study was significant because it allowed all participating laboratories to use their own detection strategies to identify IS6110, and the final results clearly indicate that existing protocols are severely deficient in terms of both sensitivity and specificity.
Another study by Lee et al. (1994) reported false positives of 62% while analyzing cerebrospinal fluid samples obtained from patients with tubercular meningitis. While specimen contamination from amplified IS6110 DNA originating from previously processed samples in the same laboratory may explain some false positives, this is not a major source of error because most laboratories maintain excellent specimen containment procedures to avoid contamination. This large number of false positive is because of the occurrence of IS6110 like sequences in organisms other than M. tuberculosis. IS6110 is a transposable insertion element (Calos and Miller), and these fragments of DNA have the property of being “mobile”. IS6110 is also likely to have originated from (or been passed on to) other organisms, and certain regions of DNA may have remained conserved among these organisms during evolution. A report published by Mariani et al. also discusses the horizontal transfer between organisms of sequences related to the M. tuberculosis IS6110 element. This would explain some of the false positive tests reported in the literature. Additionally, Kent et al. were able to amplify sequences related to IS6110 from mycobacteria other than M. tuberculosis, confirming the suspicion that IS6110 like sequences were present in other organisms, and that they could be detected in a PCR carried out with IS6110 specific primers designed to detect M. tuberculosis. In order to address this issue, a systematic analysis of nucleic acid sequences deposited in GenBank was carried out and it was found stretches of sequences similar to IS6110 in organisms other than M. tuberculosis. Many of these organisms are found in clinical specimens.
Another fact which makes IS6110 an unsuitable target for the detection of tuberculosis is that some recent reports has showed that some M. tuberculosis isolates may altogether lack IS6110 sequence in its genome thus leading to false negative results. Studies on Asian isolates have reported that this sequence may be missing in at least some of the isolates (Yuen, L. K, et al. 1993) (Yuen, L. K. w. Ross. B. C, Jackson. K. M. and Dwyer. B. 1993. J. Clin. Microbiol. 31: 1615-1618.).
Another very important aspect of detection, differentiation and treatment of tuberculosis is the emergence of human immuno deficiency virus (HIV). Epidemiology and etiology of tuberculosis has undergone change since the rise of HIV, the causative agent for acquired immuno deficiency syndrome (ADS). Incidence of tuberculosis has increased considerably since the emergence of AIDS (Bafica, et al. 2003) Among AIDS deaths over 30% are due to tuberculosis. Since 1991 number of tuberculosis patients infected with HIV has increased from 3% to over 10%. Among AIDS patients only M. tuberculosis and M. bovis are not the only causative agents for tuberculosis. So called non-tuberculous mycobacteria have become significant pathogens in immunocompromised tuberculosis patients. Different laboratories have isolated other pathogenic mycobacteria called as non-tuberculous mycobacteria from clinical specimens derived from patients co-infected with HIV. Most important among them are M. avium and closely related group of mycobacteria i.e. M. intracellulare and M. chelonae. These organisms are known as Mycobacterium avium-intracellulare complex (MM complex) organisms. MAI complex of organisms presents symptoms that are indistinguishable from tuberculosis. They are responsible for pulmonary as well as disseminated form of disease in a large number of patients especially those infected with human immuno-deficiency virus (HIV). M. avium alone has been isolated from up to 30% of clinical specimens from pulmonary tuberculosis patients and at even higher number from disseminated tuberculosis patients. M. kansasii and M. scrofulaceum are other non-tuberculous mycobacteria that have been isolated from considerable number of AIDS patients with tuberculosis. Other non-tuberculosis mycobacteria are also being isolated from clinical specimen derived from AIDS patient. Reason for fewer isolation of non-tuberculosis mycobacteria may be non availability of simple, accurate and reliable tests to isolate and differentiate different types of non-tuberculosis mycobacteria. These findings suggest that the non-tuberculosis mycobacteria have become significant etiological agents in the wake of emergence of AIDS.
IS6110 is not specific for M. tuberculosis and may be absent in many isolates and other non-tubercular mycobacteria are the causative agent for tuberculosis especially in patients co-infected with HIV. It is clear from published reports that no existing technique based on IS6110 and other target sequence provides a level of confidence needed in a clinical diagnostic test.
This accentuates need for change in the approach of detection of tuberculosis. This calls for evaluation of new targets that are able to detect all pathogenic mycobacteria in a clinical specimen instead of detecting only M. tuberculosis complex group of organisms. Ideally there should be a diagnostic method that instead of detecting only M. tuberculosis complex group of bacteria should detect all pathogenic mycobacteria including non-tubercular mycobacteria in a clinical specimen. After detection of different pathogenic mycobacteria in a clinical specimen different types of pathogenic mycobacteria can be differentiated into different species of mycobacteria by PCR-RPLF method as described in this assay. Those patients infected with NTM alone or NTM together with M. tuberculosis complex group of organism will give a quick reference for possible co-infection with HIV and thus could be a good parameter to access HIV infection and spread in the population. Not many such tests are available that can detect pathogenic mycobacteria in a clinical specimens as well as differentiate them.
Mycobacterium tuberculosis Direct Test has been evaluated by Abe et al and Miller et al. This test amplifies M. tuberculosis M. tuberculosis 16S ribosomal RNA from respiratory specimens and uses a chemiluminescent probe to detect the amplified product with a reported sensitivity of about 91%. This test is complex, takes over 24 hours to complete and uses probes to identify different mycobacteria vary only in few bases which yields false positive result if done in even slightly less stringent condition.
Success of a PCR based assay depends on several factors. Most important among them are extraction of good quality nucleic acid amenable to PCR, design of a PCR primer specific for the pathogen and a PCR condition that can specifically amplify the target sequence from the isolated DNA.
A major weakness of currently available PCR based assays for detection of mycobacteria is the lack of a method of nucleic acid extraction that is simple, efficient and ensures safety to the user. Lysis of mycobacteria and purification of nucleic acid from clinical specimen without co-purifying impurities, which are known to be present in most clinical specimens, is a crucial step in a PCR based assay. A major drawback of the published protocols is that most methods used for extracting nucleic acids cannot be easily used for all types of specimens. Any nucleic acid extraction that necessitates a tedious and inefficient DNA purification will decrease the speed and sensitivity of the test. Additionally, having to carry out a different extraction procedure on different types of samples also makes the whole process expensive and slow. Operator safety is also a major concern when handling samples containing live M. tuberculosis. It was found that after careful analysis of different DNA extraction procedures described earlier that they were either highly inefficient or unable to remove impurities that are generally present in most clinical specimens (Boom, et. al. 1990) A simple, efficient and robust method of nucleic acid extraction from various clinical specimens was thus required to ensure sensitivity and reproducibility of a PCR based assay.
Specific and non-specific amplification of the target sequence is the another crucial factor in a success PCR based assay. At the slightest of sub-optimal condition even specific and unique primers can result into nonspecific amplification of the correctly sized band and thus may lead to false positive results (Gurpreet, S et,al). This has to be addressed in a practical and cost effective way.